X-ray crystallography is a technique that is used to determine the three-dimensional arrangement of atoms within protein molecules. The x-ray diffraction pattern of a protein crystal is analyzed to determine the three dimensional structure of the protein. The growth of protein crystals is a trial and error process. Often hundreds or thousands of experiments are carried out in order to determine conditions that promote the nucleation and growth of crystals of a particular protein. Most crystallization experiments are set up in plastic trays that contain many wells.
Proteins that exist, in vivo, embedded within a cell membrane are referred to as “membrane proteins”. Examples of membrane proteins include ion channels and G-protein coupled receptors. Many membrane proteins are high-value drug targets for the pharmaceutical industry, and their three-dimensional structures would offer huge potential for the purpose of structure-based drug discovery and design. Unfortunately membrane proteins are difficult to crystallize, and as a consequence only approximately 30 high-resolution structures of different membrane proteins are currently available.
Traditionally, membrane protein crystals have been grown in solution from detergent/protein complexes. Matrices (e.g., silica gels and agarose gels) have also been used for the generation of crystals for x-ray diffraction experiments (Garcia-Ruiz, J. M., et al, “Teaching Protein Crystallization by the Gel Acupuncture Technique,” J. Chem. Edu. 75:442-446 (1998); Biertupfel, C., et al., “Crystallization of Biological Macromolecules Using Agarose Gel,” Acta Cryst. D58:1657-1659 (2002)). In such matrix-based experiments the protein is embedded in the gel-like material and a crystallization inducing agent is added. The gel reduces convection, in effect generating conditions that are close to crystallization at zero gravity, and in some cases provides a favorable environment for nucleation and growth of crystals (Rummel, G., et al., “Lipidic Cubic Phases: New Matrices for the Three-Dimensional Crystallization of Membrane Proteins,” J. Struct. Biol. 121:1-11 (1998)). One such matrix is the so-called lipid cubic phase (LCP). The use of lipidic cubic phases as a crystallization matrix has provided high-quality crystals of several membrane proteins that could not be crystallized as detergent/protein complexes (Caffrey, M., “Membrane Protein Crystallization,” J. Struct. Biol. 142(1):108-32 (2003)). Several soluble proteins have also been crystallized in an LCP matrix, suggesting that some soluble proteins that resisted crystallization using other crystallization methods may crystallize in an LCP matrix. So far, all membrane proteins crystallized in the lipidic cubic phase were colored (Chiu, M. L., et al., “Crystallization In Cubo: General Applicability to Membrane Proteins,” Acta Crystallographica D 56:781-784 (2000)).
Protein crystallization experiments are usually carried out in containers (e.g., 96 well plastic crystallization trays) that permit physical contact between the protein matrix or solution with a crystallization inducing agent (a solution or a solid). The outcome of the experiment, “crystal” or “no crystal”, is typically assessed by an operator via visual inspection using a microscope. For this purpose dissecting microscopes with bright field illumination, dark field illumination and polarization options are generally used. More advanced systems can automatically capture images of the experiments and store these in a database. The annotation of the experiment and the decision regarding the final result requires input from a human operator. Since this manual annotation is slow it creates a serious bottleneck when large numbers of crystallization setups are to be evaluated. Several automated systems have been devised to decrease the number of images that need to be inspected by the operator (Rupp, B., “High-Throughput Crystallography at an Affordable Cost: The TB Structural Genomics Consortium Crystallization Facility,” Acc. Chem. Res. 36(3):173-81. 2003; Gester, T. E., et al., “Method for Acquiring, Storing and Analyzing Crystal Images”, U.S. Pat. No. 6,529,612, 2003). These methods often carry out simple pre-selections (e.g., ignoring experiments where no changes have occurred throughout the experiment). In addition to such negative-selection procedures, image processing routines have been employed in order to identify crystals (Rupp, 2003, supra; Gester et al., 2003, supra). In virtually all cases these algorithms are based on edge detection algorithms and pattern recognition. Generally, these crystal scoring tools work well for clear-cut results and face challenges when the crystallization experiment contains small crystals, when the matrix is not perfectly transparent, or when precipitation occurs.
A number of problems are associated with crystallization of colorless membrane proteins in lipidic cubic phases. For example, low contrast due to the small difference in refractive indices of protein crystals and the surrounding lipidic phases (Nollert, P., “Microscope Detection Options for Colorless Protein Crystals Grown in Lipidic Cubic Phases,” J. Appl. Cryst. 36(5). 2003); and an obscured matrix that causes poor visibility. This may be due to the formation of protein precipitate or due to lipid that may undergo a transition into non-transparent phases during the course of crystallization. Also, crystals may be very small and difficult to see.
The foregoing problems limit the applicability of the LCP-technology for colorless membrane proteins. The present patent application provides systems and methods that use the phenomenon of birefringence to facilitate detection of crystals and enhance their contrast within a matrix. The present invention can be applied to all crystallization experiments.